# load library
library(Seurat)
library(tidyverse)
library(RCurl)
library(cowplot)
library(RColorBrewer)
library(scater)
library(dplyr)
library(SingleR)
library(celldex)

Load Data

seurat_integrated <- readRDS("results/integrated_seurat_singlets.rds")
DimPlot(seurat_integrated, reduction = "umap", label = TRUE, pt.size = .1)

Cell annotation using SingleR

mo_ref <- celldex::MouseRNAseqData()
cell_cluster <- SingleR(test = as.SingleCellExperiment(seurat_integrated),ref = mo_ref, labels = mo_ref$label.main)

# Display the counts of cells in each pruned label category
table(cell_cluster$pruned.labels)
## 
##        Astrocytes Endothelial cells  Epithelial cells       Fibroblasts 
##               215                21                 3                45 
##         Microglia           Neurons  Oligodendrocytes           T cells 
##               219             11540              2922                 1
# Assign the cell_cluster labels to the seurat_integrated object
seurat_integrated$cell_cluster <- cell_cluster$pruned.labels
# Create a dimensional plot using UMAP reduction, grouping by 'cell_cluster' with labels
DimPlot(seurat_integrated, reduction = "umap", group.by = 'cell_cluster', label = T)

# Check the length of active.ident in seurat_integrated
# This gives the number of cells after removing 'NA' cell cluster labels

seurat_integrated <- subset(seurat_integrated, cell_cluster != 'NA')

length(seurat_integrated@active.ident)
## [1] 14966
# save

Only the nuclei that matched the cell class identity were retained

Final cells used for all the downstream analysis

seu_obj <- readRDS("./results_inuse/clean_seu_obj_close.rds")

DimPlot(seu_obj, group.by= 'major_cluster',pt.size = 0.1, label = T, label.size = 3)+ NoLegend()

# Check the length of active.ident in seu_obj
# This gives the number of cells in seu_obj

length(seu_obj@active.ident)
## [1] 10995
# save
DimPlot(seu_obj, pt.size = 0.1, label = F, label.size = 3)

  1. Center for Integrative Brain Research, Seattle Children’s Research Institute, 1900 9th Ave, Seattle, WA, USA, ↩︎